We are continuing to make progress in our goal of defining germline modifiers of tumor progression and metastasis in prostate cancer. First, we are utilizing the well characterized C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive neuroendocrine prostate cancer to investigate the role of hereditary factors in the development of aggressive disease with associated neuroendocrine differentiation (NED). Our earlier work conclusively proved that the introduction of germline polymorphism into this model by breeding significantly modulates tumor progression and metastasis. Specifically, by crossing TRAMP mice to the eight progenitor strains of the Collaborative Cross (CC) recombinant inbred panel we observed profound differences in tumor burden and metastasis frequencies in several TRAMP x CC progenitor F1 strains. In the last year, we have continued to use a modifier mapping approach to identify loci driving susceptibility to aggressive disease development in the TRAMP mouse model. Our approach centers on generating F2 intercross mapping populations using the TRAMP mouse and those CC progenitor strains that displayed the greatest phenotypic variation at the F1 generation. Additionally, we are performing high resolution mapping using TRAMP x Diversity Outbred (DO) F1 males. Regarding our TRAMPxCC progenitor F2 intercross experiments, three strains were chosen for these analyses: NOD/ShiLtJ, PWK/PhJ and WSB/EiJ. We have identified multiple loci driving susceptibility to aggressive disease development in these three crosses. However, the majority of our ongoing efforts to identify candidate modifier genes are focused on the TRAMPxWSB/EiJ F2 cross. We are concentrating on this cross since the majority of aggressive disease susceptibility quantitative trait loci (QTLs) were observed in these mice. Candidate genes are being nominated using expression QTL analysis, which will define the influence of the germline on gene expression in primary tumors derived from TRAMPxWSB/EiJ F2 mice. These F2 intercross experiments are complemented by our TRAMPxDO F1 cross, which utilizes the highly genetically diverse DO heterogeneous stock mapping population. Breeding and aging of these mice has been ongoing over the last year and we anticipate to have accrued phenotype and genotype data for over 300 F1 mice by the end of 2013. We are also in the process of developing novel mouse models of prostate cancer since the molecular and histological features of TRAMP tumors are rather different from those observed in human prostate cancer. The ongoing aim here is to develop an animal model that more faithfully recapitulates the molecular events that lead to the initiation of tumorigenesis in human prostate cancer. Specifically, transgenic mice are being generated that over-expresses various combinations of oncogenes and micro-RNAs (miRNAs) targeting genes that are dysregulated in the early stages of prostate tumorigenesis. We have generated four transgenic mouse strains expressing a variety of miRNAs and oncogenes driven from prostate-specific promoters. Aging and characterization of these strains is ongoing, with the aim being to identify the transgenic strain displaying the most robust tumorigenesis and metastasis. Additionally, we are generating transgenic strains in a variety of genetic backgrounds to account for strain-specific differences in tumorigenesis. Once said strain is identified, we plan to perform the same QTL mapping experiments as were done with the TRAMP mouse using the DO and CC advanced mapping panels.